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DNA barcoding parasite organisms found in terrestrial mammal scat using COI sequence data
BIOS 35502: Practicum in Environmental Field Biology Derryl Miller
Advisor: Dr. Andy Mahon 2009
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ABSTRACT
While invasive techniques such as trapping or marking animals for study
are a concern for the safety and preservation of established ecosystems, molecular
techniques have provided accurate information about population dynamics and
the interaction between many species within an ecosystem, oftentimes without
unnecessary human interaction. Molecular techniques, involving the sequencing
of mitochondrial DNA such as the Cytochrome c Oxidase subunit I (COI), are
extremely versatile in the study of invasive, endangered, and evasive or dangerous
species. This is possible because the COI is a relatively short strand of DNA
which can be found in scat, hair, tissue, and virtually anywhere where target
organisms shed cells. This study focuses on the barcoding of parasites found in
the scat of terrestrial mammals at the University of Notre Dame Environmental
Research Center (UNDERC). From the sequencing and analysis of numerous
parasites and the generation of a phylogenic tree, this work finds that a fragment
of the COI gene is effective for the analysis of both identity of a specimen and the
relatedness between species of parasites (Bayesian statistic > 0.5 for numerous
groups) at UNDERC with the help of the National Center for Biotechnology
Information (NCBI) database.
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INTRODUCTION
Two of the largest concerns in ecology are the discovery and preservation
of biodiversity around the world (Colwell and Codington 1994). Within the study
of biodiversity of organismal groups, many correlations have been found between
a species’ size, population density, and ability to travel passively and actively to
new areas with their level of overall diversity across a region (Martiny et. al.
2006). As this broad and lofty goal, to discover and map worldwide biodiversity,
has been identified by the scientific community, several studies have made efforts
to map terrestrial and aquatic biodiversity (e.g. Colwell and Coddington 1994;
Burton and Davie 2007; Herbert et. al. 2004). Surprisingly, relatively few studies
have addressed the biodiversity of small invertebrates, interstitial organisms, or
microorganisms. This is primarily because many types and strains of these
organisms have a significantly smaller body size, often resulting in higher
population densities. Additionally, the overall trends for the genetic
diversification and extinction of these small organisms is still largely unknown
(Colwell and Coddington 1994).
Interestingly, the goal of maintaining and studying biodiversity is
interrelated with the study of small organisms because these tiny creatures often
exist within a host as parasites and can easily be detected with various DNA
extraction, amplification, and sequencing techniques described in the literature
(Monis and Andrews 2002). Furthermore, studying small parasitic organisms,
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which rely on host macroorganisms, can provide a good indication of overall
biodiversity at a study site (Monis and Andrews 2002).
There is also further interest in DNA barcoding of parasite biodiversity
because of its noninvasive nature and its capability for early detection of invasive
species, non-native species which may offset the balance of a pre-established
ecosystem where they are unfamiliar (Ficetola et. al. 2008). Catching invasive
species early, through the discovery of the introduction of new parasites into an
ecosystem, may be a useful technique for the preservation of established
ecosystems from dangerous, non-native species.
The overall purpose of this project was to utilize molecular techniques
such as DNA extraction, polymerase chain reaction (PCR), and DNA sequencing
to barcode parasite species at UNDERC, found in host mammal scat on the
property. After DNA extraction, the barcoding will be performed through DNA
amplification in the presence of universal oligonucleotide primers, specific to the
mitochondrial portions of the DNA. Barcoding of invertebrate parasites by
specifically utilizing the Cytochrome c Oxidase subunit I gene (COI) of the
mitochondrial DNA is quite common in the literature (e.g. Ficetola et. al. 2008;
Herbert et. al. 2003; Gianmarco et. al. 2009; Lunt et. al. 1996).
Cytochrome c Oxidase subunit I is an especially useful portion of DNA
because it is very diverse, capable of identifying many different species from a
smaller portion of the DNA found in the field. This gene has also been shown to
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differ about 5% between closely related species of invertebrate taxa (e.g. Mahon
et. al. 2008; Thornhill et. al. 2008). Furthermore, long-term goals of this study
include the identification and study of biodiversity and its preservation at
UNDERC.
METHODS
The purpose of this project was to provide new information as to the
diversity of the population of invertebrate parasites present at the UNDERC
property, found in host mammal scat. In order to identify parasite species at
UNDERC, these small organisms were collected from the scat of various mammal
species in an opportunistic fashion (Figure 1), wherever they could be found and
identified with the help of Elbroch’s field guide (2003). Study sites were varied
across the UNDERC property as much as possible, and GPS coordinates were
taken at the site of each scat sample (Table 1). As scat samples were recovered,
the samples were processed through 500 micron/millimeter and 125
micron/millimeter sieves, and parasites contained within the scat samples were
preserved in 95% ethanol prior to DNA extraction. After scat samples were
recovered, the DNA of each parasite specimen from the preserved samples was
extracted using a Qiagen DNEasy extraction kit according to the manufacturer’s
recommendations (Qiagen Inc., Valencia, CA).
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PCR amplification of an approximately 600 base pair fragment of the
mitochondrial COI gene was performed using universal invertebrate primers,
HCO and LCO (Folmer et. al. 1994). Individual PCR samples contained 1 μL
sample DNA to 24 μL of Master Mix, comprised of 2.5 μL TAQ Buffer, 0.5 μL
dNTP, 2.5 μL Mg2+, 0.5 μL HCO, 0.5 μL LCO, 17.35 μL H2O, and 0.15 μL TAQ.
PCR samples were incubated using COI amplification protocol (Table 3). After
PCR amplification, successful attempts were photographed with a gel camera, and
the resulting DNA was purified using a Qiagen Qiaquick PCR Gel extraction kit
according to the manufacturer’s recommendations (Qiagen Inc., Valencia, CA).
Prior to sequencing these DNA samples, a second PCR amplification was run
containing 2 μL DNA sample to 5 μL Master Mix, comprised of 1 μL TAQ
Buffer, 0.32 μL HCO or LCO, 2.68 μL H2O, and 1 μL Big Dye. The samples of
the second PCR were amplified using ABI protocol (Table 4). The resulting
DNA samples were then sequenced using an Applied Biosystems ABI3700 Gene
sequencer at the University of Notre Dame.
DNA sequences were aligned and screened using Bioedit (v7; Hall 1999)
and analyzed with a suite of molecular tools, such as Bayesian analysis, the
parsimony based TCS haplotype analysis, and sequence divergence calculations.
In order to construct a phylogenic tree, Mr. Modeltest was used to generate a
model of evolution prior to Bayesian analysis by Mr. Bayes, indicating statistical
support for individual clades within the phylogenic tree. The phylogenic tree was
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then visualized using Treeview. The sequencing of the COI in the parasites will
therefore identify individual species and, though Bayesian analysis, provide a
phylogenic tree, indicating the genetic relationship between parasite specimens
found during this study.
RESULTS
After DNA extraction, amplification, and purification, forty eight DNA
samples were sent back to Notre Dame for sequencing (Genomic sequencing
facility, Dept. of Biological Sciences). Of these forty eight DNA samples,
nineteen were successfully sequenced, and these sequences were compared to
other COI genes of morphologically similar organisms from the NCBI database.
Many of the data sequences collected at UNDERC were found to closely
resemble the COI sequences from NCBI with 80%-93% similarity in nucleotide
base pairing (Table 2).
Furthermore, the identity of many of the parasite species having a close
match of the COI gene were closely related to the parasite in question,
taxonomically. This was determined using Bioedit (v7; Hall 1999) and Bayesian
analysis, from which a phylogenic tree was constructed (Figure 2). Within the
phylogenic tree, clades of Isopoda, Hymenoptera, Coleoptera, Oligochaeta,
Nemotoda, and an Unknown order were each found to have statistically
significant genetic relationships (Bayesian statistic > 0.5) between sequence data
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of parasites found at UNDERC with the published sequences of the NCBI
database.
Our hypotheses, that sequencing and statistical comparison through
Bayesian analysis between the COI subunit of parasite specimens at UNDERC
and NCBI sequence data can accurately predict identity of parasite specimens
found in the scat of mammals, is accepted. After the construction of a phylogenic
tree, it is furthermore confirmed that the COI can accurately provide statistical
support of the relatedness between the identified invertebrate parasites found at
UNDERC.
DISCUSSION
As the ecological community continues to realize the depth of the problem
of mapping species worldwide to preserve biodiversity, molecular techniques
continue to aid in the discovery of new species, the taxonomic arrangement or
rearrangement of already discovered species, and even finding statistical evidence
of the genetic relatedness between specimens through the construction of
phylogenic trees. The COI gene fragment, being a genetically diverse and
relatively easy strand to analyze, has been a standard barcoding strand, used
around the world to discover and organize global biodiversity. In this project, the
COI gene successfully predicted the identity of parasite specimens found in the
scat of terrestrial mammals at UNDERC. Analysis of the COI also provided
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statistical evidence for the relatedness of these specimen to others found all over
the world, recorded in the NCBI.
While the Bayesian analysis of sequence data from this project did yield
statistically significant relationships (Bayesian statistic > 0.5) within clades of
Isopoda, Hymenoptera, Coleoptera, Oligochaeta, Nemotoda, and an Unknown
order, this statistical evidence was not as strong as was expected. In order to
increase the effectiveness of the Bayesian analysis, a larger sample size of
sequences with greater sequence quality, more nucleotides present per COI
sequence, is needed, but, given the financial and time constraints on this project,
finding sequence data and statistically relating these sequences to the published
sequences of the NCBI database was a success. The Unknown order is not the
discovery of a new order, but rather is an unresolved relationship containing
organisms from Diptera, Coleoptera, and Decapoda. Because of this ambiguity, it
is difficult to label the Unknown clade; however, with a larger sample size and
higher quality COI strands from DNA sequencing, this clade would likely resolve
to provide statistical support for the relationship of the sequences within this clade
to Diptera, Coleoptera, or Decapoda.
Future studies involving the sequencing of parasites and their host
macroorganisms using the COI include examining the species richness of
parasites found in scat across the property with the presence of physical barriers.
These barriers include Tenderfoot Creek or the road systems in the area, which
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may inhibit the transportation of parasite species in mammal scat, which may
cause parasite populations to differ significantly on either side of these physical
barriers. While out collecting, the majority of predator scat, coyote, wolf, and
bear, were found out in the open; however, the scat of herbivores at UNDERC,
such as deer, porcupine, and snowshoe hair were almost exclusively found in the
woods under the cover of trees. This fact might also facilitate the study of
parasites found in the scat of terrestrial predators and herbivores separately to see
if their diet or social behavior increases susceptibility to parasitism at UNDERC
(Rohde 1994). Additionally, there are many ways to track animals, including sign
and footprints; however, some sign that is discovered has been either mutilated by
the weather or is rendered unrecognizable. Another question to be answered
concerning mammal sign is whether or not enough DNA can be extracted from
mammal scat to provide genetic proof that the scat belongs to a specific mammal
species. This study would be especially useful in examining the distribution or
abundance of dangerous, evasive, or endangered mammals in an area, through
noninvasive molecular techniques (Janecka et. al. 2008; Fernandes et. al. 2008).
There are many practical applications to the field of molecular biology and
DNA barcoding. Mapping biodiversity, detecting invasive or endangered species
in an environment early to preemptively begin conservation efforts, and observing
the interaction of host and parasite species in an environment are only a few
capabilities of molecular techniques. Molecular techniques can also be
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exclusively noninvasive, which is ideal for preserving established ecosystems
while providing information as to the genetic diversity of a population without
catching or disturbing organisms in their habitat. There is an increasing potential
for conservation efforts of biodiversity worldwide as our knowledge of global
biodiversity increases, while our power to discover new genetic information with
statistical support increases significantly as online databases like the National
Center for Biotechnology Information (NCBI) continue to grow with newly
published molecular literature.
ACKNOWLEDGEMENTS
I would like to sincerely thank my research mentor, Dr. Andy Mahon, for
his excellent help and advice throughout this project. I would also like to thank
Dr. Michael Cramer, Dr. Heidi Mahon, Dr. Dave Choate, Dr. Gary Belovsky, Dr.
Jessica Hellmann, Dr. Sunny Boyd, Dr. Todd Crowl, Dr. Walt Carson, and
Andrew Perry for their continued support throughout this course in experiencing
field research. In addition, I would like to thank the University of Notre Dame
Biology Department for the opportunity to participate in Practicum in
Environmental Field Biology (UNDERC), and the entire Hank family, who
graciously continue to financially support research at UNDERC with the Bernard
J. Hank Family Endowment.
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LITERATURE CITED
Burton, T. E. and Davie, P. J. F. 2007. A revision of the shovel-nosed lobsters of
the genus Thenus (Crustacea : Decapoda : Scyllaridae), with descriptions of three new species. Zootaxa. (1429): 1-38.
Colwell, R. K.; Coddington, J. A. Estimating Terrestrial Biodiversity through
Extrapolation. 1994. Phil. Trans. R. Soc. Lond. 345:101-118. Elbroch, Mark. 2003. Mammal Tracks & Sign: A Guide to North American
Species. Stackpole Books: Pennsylvania.
Fernandes, Carlos A.; Ginja, Catarina; Pereira, Iris. 2008. Species-specific
mitochondrial DNA markers for identificationof non-invasive samples from sympatric carnivores in the Iberian Peninsula. Conserv Genet. 9:681–690.
Ficetola, G. F.; Bonini, A.; Miaud, C. 2008. Population Genetics Reveals Origin
and Number of Founders in a Biological Invasion. Molecular Ecology. 17: 773-782.
Ficetola, G. F.; Miaud, C.; Pompanon, F.; Taberlet, P. 2008. Species Detection
Using Environmental DNA from Water Samples. Biol. Lett. 4: 423-425.
Folmer, O.; Black, M.; Hoeh, W; Lutz, R.; Vrijenhoek, R. 1994. DNA primers
for amplification of mitochondrial cytochrome c oxidase subunit I from
diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3:294–299.
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Gianmarco, Ferri; Milena, Alù; Beatrice, Corradini; Manuela, Licata; Giovanni,
Beduschi. 2009. Species Identification Through DNA ‘‘Barcodes.’’ Genetic Testing and Molecular Biomarkers. 13(3): 421-426.
Herbert, P. D.; Cywinska, A.; Ball, S. L.; DeWaard, J. R. 2003. Biological
Identifications through DNA Barcodes. Proc. R. Soc. Lond. 270: 313-321. Herbert, Paul D. N.; Stoeckle, Mark Y.; Zemlak, Tyler S.; Francis, Charles M.
2004. Identification of Birds through DNA Barcodes. Plos Biology. 2(10):1657-1663.
Janecka, J. E.; Jackson, R.; Yuquang, Z.; Diqiang, L.; Munkhtsog, B.; Buckley-
Beason, V.; Murphy, W. J. 2008. Population monitoring of snow leopards using noninvasive collection of scat samples: a pilot study. Animal Conservation. 11(5): 401-411.
Lunt, D. H.; Zhang, D. X.; Szymura, J. M.; Hewitt, G. M. 1996. The insect
cytochrome oxidase I gene: Evolutionary patterns and conserved primers for phylogenetic studies. Insect Molecular Biology. 5(3):153-165.
Mahon, A. R.; Arango, C. P.; Halanych, K. M. 2008. Genetic diversity of
Nymphon (Arthropoda: Pycnogonida: Nymphonidae) along the Antarctic Peninsula with a focus on Nymphon australe. Marine Biology. 155: 315- 323.
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Martiny, J. B. H.; Bohannan, B. J. M.; Brown, J. H.; Colwell, R. K.; Fuhrman, J.
A.; Green, J. L.; Horner-Devine, M. C.; Kane, M.; Krumins, J. A.; Kuske,
C. R.; Morin, P. J.; Naeem, S.; Ovreas, L.; Reysenbah, A.; Smith, V. H.;
Staley, J. T. 2006. Microbial Biogeography; Putting Microorganisms on
the Map. Nature Publishing Group. 4: 102-112.
Monis, P. T.; Andrews, R. H.; Saint, C. P. 2002. Molecular Biology Techniques
in Parasite Ecology. International Journal for Parasitology. 32: 551-562. Rohde, K. 1994. Niche Restriction in Parasites-Proximate and Ultimate Causes.
Parasitology. 109: 69-84. Thornhill, D. J.; Mahon, A. R.; Norenberg, J. L.; Halanych, K. M. 2008. Open-
ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae). Molecular Ecology. 17(23): 5104-5117.
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TABLES
Table 1: A table relating scat location and mammal identity to the number and kinds of parasites
found within each scat sample collected in the field. Generic descriptions of the parasites found
within are included as best as they could be determined. GPS coordinates where each scat sample
was discovered are included. See Figure 1 for approximate locations on a map of the property.
Site Mammal Scat Parasites General GPS # I.D. Found Description Coordinates
1 Wolf 1 Gastropoda 16T 0304256 UTM 5124255
2 Coyote 0 16T 0304122 UTM 5124453
3 Coyote 0 16T 0304269 UTM 5125023
4 Coyote 0 16T 0304333 UTM 5125155
5 Coyote 1 Gastropoda 16T 0305019 UTM 5125673
6 Coyote 2 2 2
Gastropoda Coleoptera Unknown
16T 0305837 UTM 5125439
7 Coyote 1 1 8
Gastropoda Oligochaeta Unknown
16T 0305965 UTM 5125622
8 Wolf 9 Gastropoda 16T 0306006 UTM 5125649
9 Wolf 1 1 2 3
Gastropoda Oligochaeta Coleoptera Unknown
16T 0306072 UTM 5125671
10 Coyote 1 1
Gastropoda Coleoptera
16T 0306123 UTM 5125720
11 Coyote 1 1
Gastropoda Chilopoda
16T 0306128 UTM 5125720
12 Unable to Identify 1 Gastropoda 16T 0306249 UTM 5125847
13 Coyote 3 Gastropoda 16T 0306594 UTM 5125885
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14 Coyote 1 Gastropoda 16T 0306728 UTM 5125803
15 Coyote 3 6 6
Gastropoda Coleoptera Unknown
16T 0306805 UTM 5125793
16 Wolf 1 Gastropoda 16T 0306959 UTM 5125762
17 Coyote 2 2
Gastropoda Unknown
16T 0307268 UTM 5125578
18 Coyote 0 16T 0307665 UTM 5125621
19 Deer 1 1
Coleoptera Unknown
16T 0307601 UTM 5125727
20 Porcupine 1 1
Coleoptera Unknown
16T 0307595 UTM 5125815
21 Snowshoe Hair 0 16T 0307358 UTM 5125757
22 Coyote 2 Gastropoda 16T 0306025 UTM 5125666
23 Bear 0 16T 0305415 UTM 5122211
24 Deer 2 Coleopteraa 16T 0305361 UTM 5122155
25 Coyote 1 Hymenoptera 16T 0304023 UTM 5123635
26 Coyote 6 Unknown 16T 0304017 UTM 5123609
27 Wolf 1 6 3 4
Coleoptera Nematoda Unknown Unknown
16T 0304003 UTM 5123570
28 Coyote 1 1 1
Unknown Unknown Unknown
16T 0304001 UTM 5123565
29 Coyote 2 1 6
Coleoptera Hymenoptera
Unknown
16T 0303948 UTM 5123516
30 Mouse (Peromiscus Maniculatus)
0
31 Coyote 9 Unknown 16T 0303489 UTM 5123508
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32 Wolf 1 1
Coleoptera Unknown
16T 0303415 UTM 5123577
33 Coyote 2 Coleoptera 16T 0302753 UTM 5123787
34 Coyote 1 Unknown 16T 0302733 UTM 5123837
35 Coyote 0 16T 0302702 UTM 5123714
36 Deer 1 Unknown 37 Deer 7
1 3 4
Coleoptera Coleopteran
Larvae Unknown Unknown
16T 307600 UTM 5128981
38 Porcupine 5 1
Unknown Unknown
16T 307408 UTM 5122091
39 Porcupine 0 16T 307058 UTM 5122168
40 Coyote 0 16T 307058 UTM 5122168
41 Porcupine 1 Unknown 16T 302702 UTM 5125094
42 Porcupine 3 8
Nematoda Unknown
16T 302702 UTM 5125094
43 Deer 25 12 15 14
Dipteran Larvae Coleopteran Nematodes Unknown
16T 302780 UTM 5125038
44 Bear 25 1 5
Dipteran Larvae Hymenoptera
Unknown
16T 0303013 UTM 5125054
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Table 2: A table listing the parasites with successfully sequenced COI genes during this project
and the percentage of similarity found between the sequenced COI gene and its closest relative,
found with the National Center for Biotechnology Information (NCBI) database. Successful
parasite sequence names in the phylogenic tree (Figure 2) are also included.
Site Morphological Order Closest Relative Names % Overlap # of Parasite from NCBI in Figure 2 of COIs
1 Isopoda Nothing H01 0% 7 Coleoptera Raymunida erythrina G04 84% 7 Oligochaeta Dendrobaena octaedra E04 92% 7 Coleoptera Nothing G01 0% 16 Gastropoda Nothing F04 0% 25 Hymenoptera Camponotus
pennsylvanicus H03 89%
27 Isopoda Patelloa sp. F03 88% 27 Diptera Belvosia sp. E03 89% 27 Diptera Onthophagus clypeatus E06 91% 32 Isopoda Drosophila moriwakii D05 81% 32 Isopoda Nothing D02 0% 41 Lepidoptera Bembecia himmighoffeni C01 83% 41 Lepidoptera Quadrus contubernalis C04 92% 42 Hymenoptera Myrmecocystus melliger D04 81% 43 Coleoptera Drosophila rubida B01 85% 43 Diptera Calliphora hilli H02 91% 44 Diptera Biopyrellia bipuncta F02 91% 44 Diptera Neomyia coeruleifrons F05 93% 44 Hymenoptera Schinia pulchripennis E05 86%
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Table3: A description of COI PCR amplification protocol, including incubating temperatures and
time intervals for the Cytochrome c Oxidase subunit I prior to purification. Step 1 is a
denaturization phase. Steps 2-4 are repeated thirty times while amplification occurs. Step 5 is an
extension phase. Step 6 is a refrigeration step to preserve DNA after PCR is complete. Samples
are removed from the Thermocycler and refrigerated during step 6. Total time is about
1 hour and 40 minutes.
Steps Incubating Temperature (OC) Time Intervals 1 94 1.00 minute 2 94 30 seconds 3 50 45 seconds 4 72 1.00 minute 5 72 8.00 minute 6 4 Until Refrigerated
Table 4: A description of ABI PCR protocol, including incubating temperatures and time intervals
for the Cytochrome c Oxidase sequence I prior to sequencing. Step 1 is a denaturization phase.
Steps 2-4 are repeated 44 times while DNA amplification occurs. Step 5 is a refrigeration step to
preserve DNA after PCR is complete. Samples may be removed from the Thermocycler anytime
during step 5. This protocol is run twice for each DNA sample, using HCO for the first run and
LCO for the second run. Total time is about 3 hours and 46 minutes per run.
Steps Incubating Temperature (OC) Time Intervals 1 96 5.00 minutes 2 96 10 seconds 3 50 5 seconds 4 60 4.00 minutes 5 4 Until Refrigerated
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FIGURES
1
23
45 18
20
67 8 9
1011 12
1314 15
16
17
192122
36
24
30
23
262728
29
3132
3334
35
25
3738
3940
University of Notre Dame Environmental Research Center (UNDERC)
**39 and 40 =same GPS**Samples 41‐44 are approximate. ***41 and 42 = same GPS
41
44
4342
**
***
Figure 1: The sites of scat collection across the UNDERC property are labeled on the property
map. Refer to Table 1 for information on individual sites as well as GPS coordinates on each site.
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Isopoda
Unknown
Hymenoptera
Coleoptera
Nematoda
Phylogenic Tree
Oligochaeta
Figure 2: A phylogenic tree associating COI sequences from parasite specimen found at UNDERC
with other COI sequences found on the National Center for Biotechnology Information (NCBI)
database. Clades of Oligochaeta, Nemotoda, Coleoptera, Hymenoptera, Isopoda, and an Unknown
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21
order were each found within this phylogenic tree to have a statistically acceptable genetic
relatedness (Bayesian Statistic > 0.5) based solely on the COI sequence. Only Bayesian statistics
> 0.5 remain on this figure.